EP0167954A2 - 1-Hydroxy-cytorhodines, a microbiological method for their preparation and their use as cytostatic agents - Google Patents
1-Hydroxy-cytorhodines, a microbiological method for their preparation and their use as cytostatic agents Download PDFInfo
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- EP0167954A2 EP0167954A2 EP85108137A EP85108137A EP0167954A2 EP 0167954 A2 EP0167954 A2 EP 0167954A2 EP 85108137 A EP85108137 A EP 85108137A EP 85108137 A EP85108137 A EP 85108137A EP 0167954 A2 EP0167954 A2 EP 0167954A2
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- STUJMJDONFVTGM-CEPMKHARSA-N methyl (1r,2r,4s)-4-[4-(dimethylamino)-5-[4-hydroxy-6-methyl-5-(6-methyl-5-oxooxan-2-yl)oxyoxan-2-yl]oxy-6-methyloxan-2-yl]oxy-2-ethyl-2,5,7,10-tetrahydroxy-6,11-dioxo-3,4-dihydro-1h-tetracene-1-carboxylate Chemical compound O([C@H]1C[C@]([C@@H](C2=CC=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C(O)=C21)C(=O)OC)(O)CC)C(OC1C)CC(N(C)C)C1OC(OC1C)CC(O)C1OC1CCC(=O)C(C)O1 STUJMJDONFVTGM-CEPMKHARSA-N 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
Definitions
- the present invention further relates to a process for the preparation of compounds of formula I, which is characterized in that the strain Streptomyces purpurascens-DSM 2658 at a pH of 6.5-8.5, a temperature of 24-40 ° C and a high aeration rate are fermented in a suitable nutrient medium and the compounds of the formula I are isolated from the culture fluid and the mycelium by customary methods.
- ⁇ -Pyrromycine eg Cinerubin A (W. Keller-Schierleln, Antimicrob. Agents Chemother. 68 (1970)), various rhodirubins (H. Umezawa et al., J. Antib. 30, 6 1 6 ( 1977 ) and the aclacinomycins A 2 , B 2 , M 2 , S 2 and T 2 (T. Oki et al., J. Antib. 28, 830 (1975) and 32, 801 (1979), as well as Musetta-, Marcello- , Rudolphomycin and the like (DE Nettleton et al., J. Nat. Prod. 43, 242 (1980)).
- the process according to the invention for the preparation of compounds of the general formula I is carried out by fermentation of Streptomyces purpurascens-DSM 2658 at a pH from 6.5 - 8.5 and a temperature of 24-40 ° C under aerobic conditions with a sufficiently high oxygen supply, e.g. through a high aeration rate in a carbon and nitrogen source as well as nutrient medium containing inorganic nutrient salts and trace elements and subsequent isolation of the compounds from the Culture fluid and the mycelium carried out by conventional methods as described below.
- Glucose, starch, dextrin and glycerin are suitable as carbon sources.
- Suitable nitrogen sources are soy flour, yeast extract, meat extract, malt extract, corn steep liquor, peptone or casein.
- Suitable inorganic nutrient salts are e.g. Sodium chloride, magnesium sulfate or calcium carbonate. Iron, magnesium, copper, zinc and cobalt can be used as trace elements.
- Streptomyces purpurascens - DSM 2658 can be fermented at temperatures of 24-40 ° C with a pH of 6.5 - 8.5 and an aeration rate of 0.8 - 1.2 vvm.
- the fermentation of the Streptomyces purpurascens - DSM 2658 is preferably carried out under aerobic conditions at 30 ° C. and at a pH of 7.0. After 70-130, preferably 90-110 hours, when the highest yield is reached, the fermentation is stopped.
- the fermentation can preferably be a submerged fermentation.
- the progress of the fermentation and the formation of the anthracycline compounds can be seen from the antibacterial activity against S.aureus 209 P and Bac. subt. as well as directly by extracting the entire culture solution (mycelium and culture filtrate) with an organic solvent and measuring the absorption intensity of the violet compounds at 495, 525 and 568 nm.
- Ethyl acetate is preferably used as the organic solvent.
- the anthracycline compounds in the mycelium are extracted with an organic solvent, preferably with aqueous acetone, which has been adjusted to a pH of 3.5. After removal of the acetone, the pH of the aqueous phase is adjusted to 7.5 and then extracted at pH 7.5 with an organic solvent such as butyl acetate, ethyl acetate or chloroform, preferably with ethyl acetate.
- an organic solvent such as butyl acetate, ethyl acetate or chloroform, preferably with ethyl acetate.
- the ethyl acetate extracts from the mycelium and the culture filtrate are combined or worked up separately, concentrated and taken up in an organic solvent such as benzene or toluene. Toluene is preferably used.
- the toluene solution is then extracted with an acetate buffer with a pH of 3.5.
- DIE s er stage dividing the mixture of anthracycline derivatives into two fractions.
- fraction A the glycoside mixture remaining in the aqueous phase is referred to as fraction B.
- Fraction B is further purified according to Diagram II. As shown in Diagram II, 1-hydroxy-cytorhodins A, B, C and P and the mixture N + 0 (1: 1) are obtained from Fraction B. Fractions I, II, III and IV are undissolved mixtures of anthracycline compounds of the formula I.
- the anthracycline compounds according to the invention are distinguished by a strong action against gram-positive bacteria and a pronounced cytostatic action.
- Streptomyces purpurascens - DSM 2658 was placed on yeast-malt agar of the following compositions:
- the medium was distributed on Roux bottles and 30
- the fermentation time was 90-110 hours.
- the formation of the absorption maximum at 568 nm was followed to determine the time of harvest.
- samples of the culture solution were extracted with ethyl acetate and the absorption spectrum between 400 and 600 nm was recorded.
- Streptomyces purpurascens - DMS 2658 was grown in 2,000 ml Erlenmeyer flasks with 500 ml preculture medium and, after 2 days, inoculated with 20 1 preculture medium in a 30 l fermenter. The fermentation took place at 28 ° C. with stirring at 160-180 rpm with an aeration rate of 6-7 l / min. After 48 hours, this preculture served as an inoculum for a 300 1 fermenter with 200 1 main culture medium (Example 1). The fermentation was carried out for 90-110 hours at 28 ° C, a peripheral stirrer speed of 3.5 m / sec. and an aeration rate of 1 vvm (200 ml / min). The harvest criteria correspond to those in Example 1.
- the mycelium was separated using the usual methods (solids separator, filter press) and washed several times with a little water.
- the separated mycelia (approximately 3.4 kg) was triturated 3 times with 15 1 of acetone, concentrated acetone solution in the decanted Va k uum, with 5 1 of a mixed Na-acetate buffer solution of pH 3.5 and 3 times with 2 , 5 1 toluene washed.
- the combined ethyl acetate extracts were concentrated in vacuo.
- the separation was monitored with two flow photometers connected in series at the wavelengths 490 and 560 nm. After summary and customary processing of the red and violet fractions, the following result was achieved.
- a total of 73 mg of 1-hydroxy-cytorhodin N + 0 were obtained from two similar chromatographic runs and re-chromatographed in the above manner. 18 mg of 1-hydroxy-cytorhodin N + 0 with less than 10% residual cytorhodin N + 0 were obtained.
- a very violet product was obtained by conventional column chromatography on 31 ⁇ silica gel pH 7.5, the main component of which showed a slightly higher retention than 1-hydroxy-cytorhodin C in TLC (system A) and analytical HPLC on silica gel.
- the new compound was isolated by repeated preparative "reversed phase” HPLC.
- 175 mg of the violet crude product were dissolved in 2.5 ml of the mobile phase CHCl 3 -methanol 15% ammonium acetate in water 500: 1100: 300, and in about 100 g of 10u LiChrosorb (R) RP-18 (Merck) in a steel column 3.2 x 25 cm under pressure at a flow of 4 ml / min. chromatographed.
- the eluate was collected in fractions of 8 ml and related fractions were pooled after evaluation by analytical "reversed-phase" HPLC with double wavelength detection at 490 and 560 nm.
- silica gel 60 (Gracc) was washed with 2 N HCl 1 free of metal and, after washing out the acid in aqueous suspension, treated with 5 N NaOH until a pH of 7.5 had been established. After decanting, the modified silica gel was dried at 130 ° C., sieved and slurried into the column with the mobile phase. Solvent mixtures CHCl 3 / water / methanol 13: 4: 3-7 were used as the mobile phase. The load ratio was about 1: 100 - 1: 300.
- Silica gel plates F 254 were developed with System A: CHCl 3 -Methanol-96% acetic acid-water-triethylamine 80: 10: 10: 2: 0.01 or System B: CHCl 3 -Methanol-99% acetic acid 75: 15: 10: 2.
- the toluene phase was discarded, the aqueous phase was adjusted to pH 7.5 with 2N NaOH and extracted with 20 ml of CHC1 3 with good shaking. After drying over anhydrous Na 2 S0 4 , the mixture was filtered and evaporated in vacuo. If necessary, the residue was dissolved in a little CHC1 3 , the solution was suctioned off through a glass frit and, after adding heptane, i until turbid. V. evaporated.
- the proton resonance spectra ( 1 H-NMR spectra) were recorded on a HX-270 BRUKER Fourier transform nuclear magnetic resonance spectrometer measured at 270 MHz. The concentrations were 2-4 mg / 0.5 ml 99.8% CDCl 3 ; the solutions were shaken immediately after preparation with 0.1 ml of 5% Na 2 CO 3 in 99.5% D 2 0.
- the signals marked with an asterisk in the figures derive from low-molecular impurities in the% o range and from solvent residues.
- Mass spectra were measured on the MS-902 S, AEI mass spectrometer using an FAB (Fast Atom Bombardment) ion source.
- FAB Fluor Atom Bombardment
- the substances were introduced into the ion source in a matrix of thioglycerin, sometimes with the addition of ammonium chloride.
- the substance concentration was 10 - 30 mg / l; the absorption maxima are given in nm and the molar extinction coefficients (log ⁇ ).
- the cytostatic activity of the compounds described here was determined on L1210 mouse leukemia cells. The following test systems were used:
- the cells are incubated with different concentrations of in vitro after incubation Test substance determines the extent to which the cells can incorporate radioactive DNA precursors (eg C14-labeled thymidine).
- Untreated L1210 cells are subjected to the same test conditions and serve as a control. The method is briefly described below.
- L1210 cells in exponential growth phase (5 x 10 3 / ml in RPMI 1640) are incubated in a microtiter plate for 72 hours with different concentrations of the test substance (37 ° C, 5% C0 21 95% relative air humidity). Controls consist of cells that are only incubated with fresh medium. All determinations are carried out as 4-fold determinations. After 65 hours, 50 ⁇ l of C-1 14 thymidine (1.5 ⁇ c / ml) are added in order to radioactively label the DNA of the cell. After 7 hours of incubation, the cells are suctioned off, the DNA is precipitated with 5% trichloroacetic acid and washed successively with water or methanol.
- the radioactivity built into the DNA is determined after adding 5 ml of scintillation fluid.
- the results are given as the ratio of the scintillation index after incubation with the test substance in percent of the untreated control.
- the dose-response curve is determined from the measured values thus obtained and the IC 50 , ie the concentration which reduces the incorporation of radioactive thymidine by 50% compared to the control under test conditions, is determined graphically.
- the IC 50 values of the compounds described here in comparison to adriamycin (ADM) are summarized in Table 1.
- This method serves to demonstrate the influence of the test substances on the growth behavior of the cells over several generations (with a cell cycle time of 10-12 hours, approximately 14 consecutive generations are observed in the test time of 7 days).
- cytostatic substances reduce the number of colonies to be observed compared to an untreated control.
- the test is carried out as follows:
- the compounds of the invention have cytostatic activity, i.e. therapeutic effect against tumors, especially malignant tumors in animals and humans.
- the compounds and the acid addition salts can therefore be used as drugs for the treatment of tumors.
- the compounds can be administered in various ways depending on the dosage form.
- the compounds are normally administered mixed with standard pharmaceutical carriers or diluents. So you can e.g. individually or in a mixture together with carriers such as maltose or lactose or as non-toxic complexes, e.g. be administered as a deoxyribonucleic acid complex.
- a typical mode of administration is the injection of a solution of the compounds according to the invention in distilled water or in physiological saline.
- the solutions can be injected intraperitoneally, intravenously or intraarterially.
- Daily dose and unit dose can be determined from animal experiments and also from in vitro tests in such a way that the total dose, which is administered continuously or at intervals, does not exceed a predetermined range. So the total dose for one treatment is cycle about 0.5-5 mg / kg body weight. This dose can be administered in appropriate fractions over a period of 7 days. However, it is clear that specific doses for the treatment of humans or animals can be determined individually depending on the particular situation, for example age, body weight, gender, sensitivity, food, time of administration, further medication administered, patient's physical condition and severity the disease.
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Abstract
Die vorliegende Erfindung betrifft 1-Hydroxy-Cytorhodine der Formel 1
- Roa-dF-Rod, Roa-dF-CinA, Roa-dF=CinB. Roa-Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu oder Rod-Rod-Rod, worin Roa Rhodosamin, dF Desoxyfucose, Rod Rhodinose, Acu Aculose, CinA Cinerulose A und Cin B Cinerulose B bedeutet und dF = Cin B bedeutet, daß die beiden Zuckerbausteine durch eine zusätzliche Ätherbrücke neben der üblichen glykosidischen Bindung verknüpft sind.
- Die vorliegende Erfindung betrifft ferner ein Verfahren zur Herstellung von Verbindunaen der Formel das dadurch gekenzeichnet ist, daß man den Stamm Streptomyces purpurascens-DSM 2658 bei einer hohen Belüfungsrate in einem geeigneten Nährmedium fermentiert und die Verbindungen der Formel aus der Kulturflüssigkeit und dem Mvcel nach üblichen Methoden isoliert.
- Roa-dF-Rod, Roa-dF-CinA, Roa-dF = CinB. Roa-Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu or Rod-Rod-Rod, in which Roa is rhodosamine, dF deoxyfucose, Rod Rhodinose, Acu Aculose, CinA Cinerulose A and Cin B Cinerulose B means and dF = Cin B means that the two sugar units are linked by an additional ether bridge in addition to the usual glycosidic bond.
- The present invention further relates to a process for the preparation of compounds of the formula which is characterized in that the strain Streptomyces purpurascens-DSM 2658 is fermented at a high aeration rate in a suitable nutrient medium and the compounds of the formula from the culture liquid and the Mvcel by conventional methods isolated.
Die erfindungsgemäßen Verbindungen haben cytostatische Wirktsamkeit.The compounds according to the invention have cytostatic activity.
Description
Die vorliegende Erfindung betrifft 1-Hydroxy-Cytorhodine der Formel 1
- Roa-dF-Rod, Roa-dF-CinA, Roa-dF=CinB, Roa-Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu oder Rod-Rod-Rod, worin Roa Rhodosamin, dF Desoxyfucose, Rod Rhodinose, Acu Aculose, CinA Cinerulose A und Cin B Cinerulose B bedeutet und dF = Cin B bedeutet, daß die beiden Zuckerbausteine durch eine zusätzliche Ätherbrücke neben der üblichen glykosidischen Bindung verknüpft sind.
- Roa-dF-Rod, Roa-dF-CinA, Roa-dF = CinB, Roa-Rod-Rod, Roa-dF-Acu, Roa-Rod-CinA, Roa-Rod-Acu or Rod-Rod-Rod, where Roa Rhodosamine, dF deoxyfucose, Rod Rhodinose, Acu Aculose, CinA Cinerulose A and Cin B Cinerulose B means and dF = Cin B means that the two sugar units are linked by an additional ether bridge in addition to the usual glycosidic bond.
Die vorliegende Erfindung betrifft ferner ein Verfahren zur Herstellung von Verbindungen der Formel I, das dadurch gekenzeichnet ist, daß man den Stamm Streptomyces purpurascens-DSM 2658 bei einem pH-Wert von 6,5 - 8,5, einer Temperatur von 24-40°C und einer hohen Belüftungsrate in einem geeigneten Nährmedium fermentiert und die Verbindungen der Formel I aus der Kulturflüssigkeit und dem Mycel nach üblichen Methoden isoliert.The present invention further relates to a process for the preparation of compounds of formula I, which is characterized in that the strain Streptomyces purpurascens-DSM 2658 at a pH of 6.5-8.5, a temperature of 24-40 ° C and a high aeration rate are fermented in a suitable nutrient medium and the compounds of the formula I are isolated from the culture fluid and the mycelium by customary methods.
Die Isolierung und Identifizierung von Streptomyces purpurascens-DSM 2658 ist in der deutschen Patentanmeldung P 332 325.0 beschrieben. Der Mikroorganismus wurde am 24.5.1983 bei der DSM (Deutsche Sammlung von Mikroorganismen) in D-3400 Göttingen (Grisebachstr. 8) hinterlegt.The isolation and identification of Streptomyces purpurascens-DSM 2658 is described in German patent application P 332 325.0. The microorganism was deposited on May 24, 1983 with the DSM (German Collection of Microorganisms) in D-3400 Göttingen (Grisebachstr. 8).
In der genannten Patentanmeldung wird auch ein Verfahren zur Herstellung von 1-Deshydroxy-Anthracyclin-Derivaten der Formel I, den sogenannten Cytorhodinen, durch Fermentation des Stammes Streptomyces purpurascens-DSM 2658 unter aeroben Bedingungen beschrieben.In the patent application mentioned, a process for the preparation of 1-deshydroxy-anthracycline derivatives of the formula I, the so-called cytorhodins, by fermentation of the strain Streptomyces purpurascens-DSM 2658 under aerobic conditions is also described.
Es wurde nun überraschenderweise gefunden, daß bei Erhöhung der Sauerstoffversorgung bei der Fermentation 1-Hydroxy-Cytorhodine gebildet werden. Diese neuen vor allem cytostatisch wirksamen Verbindungen gehören zur Gruppe der 1-Hydroxy-Anthracycline, von denen die folgenden Vertreter bereits beschrieben sind:
- 1-Hydroxy-ß-Rhodomycine - auch ß-Iso-Rhodomycine genannt-: z.B. Violamycine A2 und B I - 1 (W. Fleck et al., Z. Allgem. Mikrobiologie 14, 551 (1974) und ß-Isorhodomycine (H. Brockmann et al., Chem. Ber. 98, 3145 (1965).
- 1-Hydroxy-ß-Rhodomycine - also called ß-Iso-Rhodomycine-: e.g. Violamycine A 2 and BI - 1 (W. Fleck et al., Z. Allgemeine. Mikrobiologie 14, 551 (1974) and ß-Isorhodomycine (H Brockmann et al., Chem. Ber. 98, 3145 (1965).
ε-Pyrromycine (Cinerubine): -z.B. Cinerubin A (W. Keller-Schierleln, Antimicrob. Agents Chemother. 68 (1970)), verschiedene Rhodirubine (H. Umezawa et al., J. Antib. 30, 616 (1977) und die Aclacinomycine A2, B2, M2, S2 und T 2 (T. Oki et al., J. Antib. 28, 830 (1975) und 32, 801 (1979), sowie Musetta-, Marcello-, Rudolphomycin u.ä. (D.E. Nettleton et al., J. Nat. Prod. 43, 242 (1980)).ε-Pyrromycine (Cinerubine): eg Cinerubin A (W. Keller-Schierleln, Antimicrob. Agents Chemother. 68 (1970)), various rhodirubins (H. Umezawa et al., J. Antib. 30, 6 1 6 ( 1977 ) and the aclacinomycins A 2 , B 2 , M 2 , S 2 and T 2 (T. Oki et al., J. Antib. 28, 830 (1975) and 32, 801 (1979), as well as Musetta-, Marcello- , Rudolphomycin and the like (DE Nettleton et al., J. Nat. Prod. 43, 242 (1980)).
Das erfindungsgemäße Verfahren zur Herstellung von Verbindungen der allgemeinen Formel I, wird durch Fermentation von Streptomyces purpurascens-DSM 2658 bei einem pH-Wert von 6,5 - 8,5 und einer Temperatur von 24-40°C unter aeroben Bedingungen bei einer genügend hohen Sauerstoffversorgung z.B. durch eine hohe Belüftungsrate in einem Kohlenstoff- und Stickstoffquellen sowie anorganische Nährsalze und Spurenelemente enthaltenden Nährmedium und anschließende Isolierung der Verbindungen aus der Kulturflüssigkeit und dem Mycel nach üblichen Methoden wie nachfolgend beschrieben, durchgeführt.The process according to the invention for the preparation of compounds of the general formula I is carried out by fermentation of Streptomyces purpurascens-DSM 2658 at a pH from 6.5 - 8.5 and a temperature of 24-40 ° C under aerobic conditions with a sufficiently high oxygen supply, e.g. through a high aeration rate in a carbon and nitrogen source as well as nutrient medium containing inorganic nutrient salts and trace elements and subsequent isolation of the compounds from the Culture fluid and the mycelium carried out by conventional methods as described below.
Als Kohlenstoffquellen eignen sich Glucose, Stärke, Dextrin und Glycerin. Geeignete Stickstoffquellen sind Sojamehl, Hefeextrakt, Fleischextrakt, Malzextrakt, Maisquellwasser, Pepton oder Casein. Als anorganische Nährsalze eigne sich z.B. Natriumchlorid, Magnesiumsulfat oder Calciumkarbonat. Als Spurenelemente kann man Eisen, Magnesium, Kupfer, Zink und Kobalt verwenden.Glucose, starch, dextrin and glycerin are suitable as carbon sources. Suitable nitrogen sources are soy flour, yeast extract, meat extract, malt extract, corn steep liquor, peptone or casein. Suitable inorganic nutrient salts are e.g. Sodium chloride, magnesium sulfate or calcium carbonate. Iron, magnesium, copper, zinc and cobalt can be used as trace elements.
Streptomyces purpurascens - DSM 2658 kann bei Temperaturen von 24-40°C bei einem pH von 6,5 - 8,5 und einer Belüftungsrate von 0,8 - 1,2 vvm fermentiert werden. Vorzugsweise erfolgt die Fermentation des Streptomyces purpurascens - DSM 2658 unter aeroben Bedingungen bei 30°C und bei einem pH-Wert von 7,0. Nach 70 - 130, vorzugsweise 90 - 110 Stunden, wenn die höchste Ausbeute erreicht ist, bricht man die Fermentation ab. Vorzugsweise kann es sich bei der Fermentation um eine Submers-Fermentation handeln.Streptomyces purpurascens - DSM 2658 can be fermented at temperatures of 24-40 ° C with a pH of 6.5 - 8.5 and an aeration rate of 0.8 - 1.2 vvm. The fermentation of the Streptomyces purpurascens - DSM 2658 is preferably carried out under aerobic conditions at 30 ° C. and at a pH of 7.0. After 70-130, preferably 90-110 hours, when the highest yield is reached, the fermentation is stopped. The fermentation can preferably be a submerged fermentation.
Der Fortschritt der Fermentation und die Bildung der Anthracyclinverbindungen kann anhand der antibakteriellen Wirksamkeit gegen S.aureus 209 P und Bac. subt. sowie direkt durch Extraktion der gesamten Kulturlösung (Mycel und Kulturfiltrat) mit einem organischen Lösungsmittel und Messung der Absorptionsintensität der violetten Verbindungen bei 495, 525 und 568 nm verfolgt werden. Als organisches Lösungsmittel verwendet man vorzugsweise Äthylacetat.The progress of the fermentation and the formation of the anthracycline compounds can be seen from the antibacterial activity against S.aureus 209 P and Bac. subt. as well as directly by extracting the entire culture solution (mycelium and culture filtrate) with an organic solvent and measuring the absorption intensity of the violet compounds at 495, 525 and 568 nm. Ethyl acetate is preferably used as the organic solvent.
Die Anthracyclinverbindungen im Kulturfiltrat und im Mycel werden gemäß dem Schema im Schaubild 1 (vgl. auch Beispiel 3) der vorliegenden Anmeldung isoliert.The anthracycline compounds in the culture filtrate and in the mycelium are isolated according to the scheme in Figure 1 (cf. also Example 3) of the present application.
Die Anthracyclinverbindungen im Mycel werden mit einem organischen Lösungsmittel, vorzugsweise mit wäßrigem Aceton, das auf einen pH-Wert von 3,5 eingestellt wurde, extrahiert. Nach Entfernung des Acetons stellt man den pH-Wert der wäßrigen Phase auf 7,5 ein und extrahiert dann bei einem pH-Wert von 7,5 mit einem organischen Lösungsmittel wie Butylacetat, Äthylacetat oder Chloroform, vorzugsweise mit Äthylacetat. Die Äthylacetatextrakte aus dem Mycel und dem Kulturfiltrat werden vereinigt oder separat aufgearbeitet, konzentriert und in einem organischen Lösungsmittel, wie Benzol oder Toluol, aufgenommen. Vorzugsweise verwendet man Toluol. Die Toluollösung extrahiert man dann mit einem Acetatpuffer von einem pH-Wert von 3,5. Auf die- ser Stufe teilt man das Gemisch der Anthracyclin-Derivate in zwei Fraktionen auf. Das in der Toluolphase verbleibende Gemisch bezeichnet man als Fraktion A, das in der wäßrigen Phase verbleibende Glykosidgemisch bezeichnet man als Fraktion B.The anthracycline compounds in the mycelium are extracted with an organic solvent, preferably with aqueous acetone, which has been adjusted to a pH of 3.5. After removal of the acetone, the pH of the aqueous phase is adjusted to 7.5 and then extracted at pH 7.5 with an organic solvent such as butyl acetate, ethyl acetate or chloroform, preferably with ethyl acetate. The ethyl acetate extracts from the mycelium and the culture filtrate are combined or worked up separately, concentrated and taken up in an organic solvent such as benzene or toluene. Toluene is preferably used. The toluene solution is then extracted with an acetate buffer with a pH of 3.5. In DIE s er stage dividing the mixture of anthracycline derivatives into two fractions. The mixture remaining in the toluene phase is referred to as fraction A, the glycoside mixture remaining in the aqueous phase is referred to as fraction B.
Die Fraktion B wird weiter gereinigt gemäß Schaubild II. Wie im Schaubild II dargestellt, erhält man aus der Fraktion B die 1-Hydroxy-Cytorhodine A, B, C und P und das Gemisch N + 0 (1:1). Bei den Fraktionen I, II, III und IV handelt es sich um nicht aufgetrennte Mischungen aus Anthracyclinverbindungen der Formel I.Fraction B is further purified according to Diagram II. As shown in Diagram II, 1-hydroxy-cytorhodins A, B, C and P and the mixture N + 0 (1: 1) are obtained from Fraction B. Fractions I, II, III and IV are undissolved mixtures of anthracycline compounds of the formula I.
Die aus der Kulturlösung von Streptomyces purpurascens - DSM 2658 isolierten und gemäß Schaubild I gereinigten Verbindungen haben die allgemeine Formel I.
Die bevorzugten erfindungsgemäßen Verbindungen haben die in Formel II
Die erfindungsgemäßen Anthracyclinverbindungen zeichnen sich durch eine starke Wirkung gegen grampositive Bakterien sowie eine ausgeprägte cytostatische Wirkung aus.The anthracycline compounds according to the invention are distinguished by a strong action against gram-positive bacteria and a pronounced cytostatic action.
Die vorliegende Erfindung wird durch die nachfolgenden Beispiele näher erläutert:The following examples illustrate the present invention:
Herstellung von 1-Hydroxy-Cytorhodinen durch Fermentation von Streptomyces purpurascens - DSM 2658.Production of 1-hydroxy-cytorhodins by fermentation of Streptomyces purpurascens - DSM 2658.
Streptomyces purpurascens - DSM 2658 wurde auf Hefe-Malz-Agar der folgenden Zusammensetzungn gegeben:
Dazu wurde das Medium auf Roux-Flaschen verteilt und 30For this purpose, the medium was distributed on Roux bottles and 30
Minuten bei 121°C sterilisiert, abgekühlt, mit einer Sporensuspension beimpft und 7 - 14 Tage bei 25°C inku- biert. Danach konnte man ein gutes Wachstum und eine gute Sporenbildung feststellen. Von dieser Stammkultur wurde eine Sporensuspension mit 107 Sporen/ml in steriler NaCl (0,8 %ig) hergestellt. 10 ml dienten als Inokulum von 500 ml folgenden Vorkulturmediums:
Je 500 ml des obigen Mediums wurden auf 2 000 ml Erlen- meyerkolben verteilt und 30 Min. bei 121°C sterilisiert. Die beimpften Kolben wurden 2 Tage bei 25°C und 200 Upm auf einem Rotationsschüttler inkubiert.500 ml of the above medium were distributed to 2 000 ml alder m eyerkolben and sterilized 30 min. At 121 ° C. The inoculated flasks were incubated on a rotary shaker at 25 ° C. and 200 rpm for 2 days.
500 ml der gewachsenen Vorkultur wurden als Inokulum für folgende Hauptkultur verwendet:
9 Liter des obigen Mediums wurden in einen 12 1-Fermenter gegeben und 50 Min. bei 121°C sterilisiert. Die Fermentation wurde bei 28°C, einer Rührergeschwidigkeit von 600 Upm und einer Belüftungsrate von 8 - 9 1 Luft/Min. durchgeführt. 9 liters of the above medium were placed in a 12 liter fermenter and sterilized for 50 minutes at 121 ° C. The fermentation was carried out at 28 ° C., a stirrer speed of 600 rpm and an aeration rate of 8-9 l air / min. carried out.
Die Fermentationsdauer betrug 90 - 110 Stunden. Zur Feststellung des Aberntezeitpunktes wurde die Ausbildung des Absorptionsmaximums bei 568 nm verfolgt. Dazu wurden Proben der Kulturlösung mit Äthylacetat extrahiert und das Absorptionsspektrum zwischen 400 und 600 nm aufgenommen.The fermentation time was 90-110 hours. The formation of the absorption maximum at 568 nm was followed to determine the time of harvest. For this purpose, samples of the culture solution were extracted with ethyl acetate and the absorption spectrum between 400 and 600 nm was recorded.
Fermentation von Streptomyces purpurascens - DSM 2658 in größerem Maßstab.Fermentation of Streptomyces purpurascens - DSM 2658 on a larger scale.
Gemäß Beispiel 1 wurde Streptomyces purpurascens - DMS 2658 in 2 000 ml-Erlenmeyerkolben mit 500 ml Vorkulturmedium angezogen und nach 2 Tagen in einen 30 1-Fermenter mit 20 1-Vorkulturmedium überimpft. Die Fermentation erfolgte bei 28°C unter Rühren bei 160 - 180 Upm mit einer Belüftungsrate von 6 - 7 l/Min. Nach 48 Stunden diente diese Vorkultur als Inokulum für einen 300 1-Fermenter mit 200 1-Hauptkulturmedium (Beispiel 1). Die Fermentation erfolgte 90 - 110 Stunden bei 28°C, einer Rührerumfangsgeschwindigkeit von 3,5 m/sec. und einer Belüftungsrate von 1 vvm (200 ml/min). Die Aberntekriterien entsprechenden denen in Beispiel 1.According to Example 1, Streptomyces purpurascens - DMS 2658 was grown in 2,000 ml Erlenmeyer flasks with 500 ml preculture medium and, after 2 days, inoculated with 20 1 preculture medium in a 30 l fermenter. The fermentation took place at 28 ° C. with stirring at 160-180 rpm with an aeration rate of 6-7 l / min. After 48 hours, this preculture served as an inoculum for a 300 1 fermenter with 200 1 main culture medium (Example 1). The fermentation was carried out for 90-110 hours at 28 ° C, a peripheral stirrer speed of 3.5 m / sec. and an aeration rate of 1 vvm (200 ml / min). The harvest criteria correspond to those in Example 1.
Isolierung des Rohgemisches der Anthracyclinverbindungen.Isolation of the raw mixture of anthracycline compounds.
190 1 KL wurden mit 0,5 % Formalin versetzt und mit Essigsäure auf pH = 5,2 gestellt. Das Mycel wurde mit den üblichen Methoden (Feststoffseparator, Filterpresse) abgetrennt und mehrmals mit wenig Wasser gewaschen.190 1 KL were mixed with 0.5% formalin and adjusted to pH = 5.2 with acetic acid. The mycelium was separated using the usual methods (solids separator, filter press) and washed several times with a little water.
Das Kulturfiltrat (ca. 185 1) wurde mit dem Waschwasser vereinigt, mit NaOH auf pH = 7,5 - 8,0 gestellt und 2 x mit je 50 1 Ethylacetat extrahiert. Die wässrige Phase wurde verworfen. Die organischen Phasen wurden vereinigt, im Vakuum auf 8 1 eingeengt und dann 4 x mit 3 1 Natriumacetat-Pufferlösung (pH 3,5) extrahiert. Die vereinigten Pufferlösungen wurden anschließend 3 x mit 3 1 Toluol gewaschen.The culture filtrate (approx. 185 l) was combined with the wash water, adjusted to pH = 7.5-8.0 with NaOH and twice extracted with 50 1 of ethyl acetate. The aqueous phase was discarded. The organic phases were combined, concentrated to 8 1 in vacuo and then extracted 4 × with 3 1 sodium acetate buffer solution (pH 3.5). The combined buffer solutions were then washed 3 times with 3 l of toluene.
Das abgetrennte Mycel (ca. 3,4 kg) wurde 3 mal mit 15 1 Aceton ausgerührt, die abdekantierte Acetonlösung im Va- kuum eingeengt, mit 5 1 einer Na-Acetat-Pufferlösung vom pH 3,5 versetzt und 3 mal mit 2,5 1 Toluol gewaschen.The separated mycelia (approximately 3.4 kg) was triturated 3 times with 15 1 of acetone, concentrated acetone solution in the decanted Va k uum, with 5 1 of a mixed Na-acetate buffer solution of pH 3.5 and 3 times with 2 , 5 1 toluene washed.
Die wässrige Pufferphase der Kulturfiltrataufarbeitung wurde mit NaOH auf pH = 7,9 gestellt und 3 x mit 3 1 Ethylacetat extrahiert. Die vereinigten Ethylacetatextrakte wurden im Vakuum eingeengt.The aqueous buffer phase of the culture filtrate work-up was adjusted to pH = 7.9 with NaOH and extracted 3 × with 3 l of ethyl acetate. The combined ethyl acetate extracts were concentrated in vacuo.
Die wässrige Pufferphase der Mycelaufarbeitung wurde mit NaOH auf pH = 8,0 gestellt und 3 x mit je 2,5 1 Ethylacetat extrahiert. Die vereinigten organischen Extrakte wurden im Vakuum eingeengt.The aqueous phase of the buffer Mycelaufarbeitung has been made and with N a OH to pH = 8.0 3 x extracted with 2.5 1 of ethyl acetate. The combined organic extracts were concentrated in vacuo.
Die verbleibenden wässrigen Phasen wurden verworfen.The remaining aqueous phases were discarded.
Isolierung von 1-Hydroxy-Cytorhodin A durch "reversed phase" HPLC.Isolation of 1-hydroxy-cytorhodin A by "reversed phase" HPLC.
Eine stark violette Charge von Cytorhodin A aus rohem polarem Cytorhodin-Gemisch (Komplex II), gewonnen durch übliche Säulenchromatogrpahie an Kieselgel mit essig- säurehaltiger mobiler Phase und nachfolgende, weitere An- reicherung durch präparative HPLC an SiO2, wurde durch präparative HPLC an einem "reversed phase" Adsorbens weiter aufgetrennt. So wurden z.B. 250 mg in 4 ml einer mobilen Phase CHCl3-Methanol-10 % Ammonacetat in Wasser 150 : 1050 : 375 gelöst und auf eine Stahlsäule (3,2 x 25 cm), mit ca. 120 g LiChrosorb(R) RP-18, 25-40 p (Merck) unter Druck gefüllt gegeben und bei einem Fluß von 4 ml/Min. chromatographiert.A strong violet batch of cytorhodin A from crude cytorhodin polar mixture (complex II), recovered by conventional Säulenchromatogrpahie on silica gel with acetic s äurehaltiger mobile phase and subsequent further check re ic h augmentation by preparative HPLC on SiO 2, was prepared by preparative HPLC separated on a "reversed phase" adsorbent. Thus, for example, 250 mg in 4 ml of a mobile phase CHCl 3 -methanol-10% ammonium acetate in
Die Trennung wurde mit zwei hintereinander geschalteten Durchflußphotometern bei den Wellenlängen 490 und 560 nm verfolgt. Nach Zusammenfassung und üblicher Aufarbeitung jeweils der roten und der violetten Fraktionen wurde folgendes Ergebnis erreicht.The separation was monitored with two flow photometers connected in series at the wavelengths 490 and 560 nm. After summary and customary processing of the red and violet fractions, the following result was achieved.
Fr. 88 - 108 62 mg Cytorhodin A (nach 1H-NMR) 109 - 120 46 mg 1-Hydroxy-Cytorhodin A (ca. 80 - 90 % Reinheit)88 - 108 62 mg cytorhodin A (according to 1 H-NMR) 109 - 120 46 mg 1-hydroxy-cytorhodin A (approx. 80 - 90% purity)
Etwa 150 mg von auf obige Weise gewonnenem 1-Hydroxy-Cytorhodin A wurde durch erneute präparative HPLC an RP-18, 25-40 µ unter obigen Bedingungen weiter gereinigt. Erhalten wurden 60 mg 1-Hydroxy-Cytorhodin A mit weniger als 2 % restlichem Cytorhodin A.
C60H88N2O21, M ber.: 1172 (FAB-MS bestätigt).C 60 H 88 N 2 O 21 , M calc .: 1172 (FAB-MS confirmed).
Isolierung von 1-Hydroxy-Cytorhodin V durch präparative Mitteldruck- und Schichtchromatographie.Isolation of 1-hydroxy-cytorhodin V by preparative medium pressure and layer chromatography.
7 g rohes Cytorhodingemisch, gewonnen durch Extraktion aus der Kulturlösung wie im Schema I beschrieben, wurden an 620 g Kieselgel 31 µ (Grace) in zwei radial komprimierten Silica-Patronen (Waters, Prep LC/System 500(R)) mit dem Laufmittelgemisch CHC13/Methanol/96%ige Essigsäure/Wasser = 80 : 10 : 10 : 2-chromatographiert. Die Probe wurde in 65 ml der mobilen Phase auf die äquilibrierte Säule gegeben und bei einem Fluß von 25 ml/Min. in Fraktionen zu 23 ml aufgetrennt. Die Beurteilung erfolgte dünnschichtchromatographisch und mit Hilfe der analytischen HPLC.7 g of crude cytorhode mixture, obtained by extraction from the culture solution as described in Scheme I, were on 620 g of 31 µ (Grace) silica gel in two radially compressed silica cartridges (Waters, Prep LC / System 500 (R) ) with the CHC1 solvent mixture 3 / methanol / 96% acetic acid / water = 80: 10: 10: 2 chromatographed. The sample was placed in 65 ml of the mobile phase on the equilibrated column and at a flow of 25 ml / min. separated into 23 ml fractions. The assessment was carried out by thin layer chromatography and with the aid of analytical HPLC.
In Fraktion 29 - 34 fielen 195 mg eines Gemisches aus Cytorhodin V und 1-Hydroxy-Cytorhodin V in einer Reinheit von 80 % an (Rf 0,38 im System A).Fractions 29-34 gave 195 mg of a mixture of cytorhodin V and 1-hydroxy-cytorhodin V in a purity of 80% (R f 0.38 in system A).
60 mg dieser Probe wurden durch präparative Dünnschichtchromatographie (Merck, DC-Fertigplatten Kieselgel 60) weiter gereinigt.60 mg of this sample were further purified by preparative thin layer chromatography (Merck, TLC ready-made silica gel 60 plates).
Die Elution der herausgeschabten Banden erfolgte mit CHC13/Methanol 1 : 1. Die Elutionslösung wurde mit wässriger Dinatriumhydrogenphosphat-Lösung neutralisiert und mit Wasser bis zur Abtrennung der CHCl3-Phase versetzt. Die Chloroformphase wurde separiert, einmal mit wenig Wasser gewaschen, über wasserfreiem Na2SO4 getrocknet und eingedampft. Nach Aufnehmen in wenig Chloroform wurde mit der 10fachen Volumenmenge Petroläther gefällt und der Niederschlag i.V. getrocknet. Es wurden neben 12 mg Cytorhodin V 17 mg 1-Hydroxy-Cytorhodin V erhalten.The bands scraped out were eluted with CHC1 3 / methanol 1: 1. The elution solution was neutralized with aqueous disodium hydrogenphosphate solution and water was added until the CHCl 3 phase had been separated off. The chloroform phase was separated, washed once with a little water, dried over anhydrous Na 2 SO 4 and evaporated. After taking up in a little chloroform, it was precipitated with 10 times the volume of petroleum ether and the precipitate was dried in vacuo. In addition to 12 mg of cytorhodin V, 17 mg of 1-hydroxy-cytorhodin V were obtained.
V: durch 1H-NMR identifiziertV : identified by 1 H-NMR
C60H84N2O20, M ber, 1152 (FAB-MS bestätigt).
C60H84N2O21, M ber.: 1168 (FAB-MS bestätigt).C 60 H 84 N 2 O 21 , M calc .: 1168 (FAB-MS confirmed).
Isolierung von 1-Hydroxy-Cytorhodin B durch "reversed phase" HPLC.Isolation of 1-hydroxy-cytorhodin B by "reversed phase" HPLC.
Eine durch übliche präparative Säulenchromatographie an Kieselgel (essigsäurehaltige mobile Phase) gewonnene violette Charge von Cytorhodin B wurde an einer mit 30 g 10 Lichrosorb(R) RP-18 (Merck) gefüllten Stahlsäule (1,6 x 25 cm) unter Druck chromatographiert. Dazu wurden 120 mg in 1 ml der mobilen Phase CHCl3-MeOH-10 % Ammonacetat in Wasser 150 : 1050 : 375 gelöst und injiziert. Bei einem Fluß von 4 ml/Min. wurden Fraktionen von 4 ml aufgefangen und durch Messung der Extinktion bei 490 und 570 nm beurteilt.A by conventional preparative column chromatography on silica gel (acetic acid-containing mobile phase) obtained violet batch of cytorhodin B was chromatographed on a filled with 30 g of 10 Lichrosorb (R) RP-18 (Merck) steel column (1.6 x 25 cm) under pressure. For this purpose, 120 mg were dissolved in 1 ml of the mobile phase CHCl 3 -MeOH-10% ammonium acetate in water 150: 1050: 375 and injected. With a flow of 4 ml / min. 4 ml fractions were collected and assessed by measuring the absorbance at 490 and 570 nm.
Zusammengehörende Fraktionen wurden vereinigt und wie üblich aufgearbeitet.Associated fractions were combined and processed as usual.
Es wurden erhalten:
B: Identifizierung durch 1H-NMR, Absorptionsspektrum und FAB-MSB: Identification by 1 H-NMR, absorption spectrum and FAB-MS
Isolierung von 1-Hydroxy-Cytorhodin C durch "reversed phase" HPLC.Isolation of 1-hydroxy-cytorhodin C by "reversed phase" HPLC.
Ein durch übliche präparative Säulenchromatographie an Kieselgel gewonnenes rohes violettes Cytorhodin C haltiges Produkt (25 mg) wurde an einer mit 30 g 10 n Lichrosorb(R) RP-18 (Merck) gefüllten Stahlsäule (1,6 x 25 cm) mit der Mischung CHC13-Methanol-10 % Ammonacetat in Wasser 150 : 1050 : 375 bei einem Fluß von 2 ml/Min. chromatographiert. Fraktionen von 2 ml wurden aufgefangen und nach Beurteilung durch Messung der Extinktionen bei 490 und 570 nm vereinigt. Nach üblicher Aufarbeitung wurden erhalten:
- Fr. 80 - 142 10 mg Cytorhodin C
- 159 - 175 7 mg 1-OH-Cytorhodin C
- Fr. 80 - 142 10 mg cytorhodin C
- 159-175 7 mg 1-OH-cytorhodin C
C60H80N2O23, M ber., 1196 (FAB-MS bestätigt).C 60 H 80 N 2 O 23 , M calc., 1196 (FAB-MS confirmed).
Isolierung von 1-Hydroxy-Cytorhodin N + 0 durch "reversed phase" - HPLC.Isolation of 1-hydroxy-cytorhodin N + 0 by "reversed phase" - HPLC.
Durch übliche mehrstufige Säulenchromatographie an 31 Kieselgel, pH 7,5, und anschließend an 10 µ LiChrosorb(R) RP-18 (s.a. Beispiel 4) wurde eine stark violette Charge eines sich nach DC und analytischer HPLC an Kieselgel mit System B wie Cytorhodin N + 0 verhaltenden Produktes gewonnen, die durch wiederholte präparative HPLC an "reversed phase"-Adsorbentien weiter in einen roten und einen violetten Anteil aufgetrennt werden konnte.By conventional multi-stage column chromatography on 31 silica gel, pH 7.5, and then on 10 .mu.LiChrosorb ( R ) RP-18 (see also Example 4), a very violet batch of a silica gel with System B such as cytorhodin N was obtained by TLC and analytical HPLC + 0 behavioral product, which could be further separated into a red and a violet portion by repeated preparative HPLC on "reversed phase" adsorbents.
Dazu wurden z.B. 230 mg des violetten Produktes in 4 ml der mobilen Phase CHCl3-Methanol-10 % Ammonacetat in Wasser 150 : 1050 : 375 gelöst und an ca. 100 g 10 µ Lichrosorb(R) RP-18 (Merck) in einer Stahlsäule 3,2 x 25 cm unter Druck bei einem Fluß von 4 ml/Min. chromatographiert. Fraktionen von 8 ml wurden gesammelt und nach Beurteilung durch analytische "reversed phase" HPLC mit Doppelwellenlängendetektion bei 490 und 560 nm zusammengefaßt. Erhalten wurden nach üblicher Aufarbeitung:
- Fr. 70 - 85 97 mg Cytorhodin N + 0 (ca. 70 - 80 % Reinheit - nach 1H-NMR)
- 86 - 95 40 mg 1-Hydroxy-Cytorhodin N + 0 (ca. 60 % Reinheit)
- Fr. 70 - 85 97 mg cytorhodin N + 0 (approx. 70 - 80% purity - according to 1 H-NMR)
- 86 - 95 40 mg 1-hydroxy-cytorhodin N + 0 (approx. 60% purity)
Aus zwei ähnlichen chromatographischen Läufen wurden zusammen 73 mg 1-Hydroxy-Cytorhodin N + 0 gewonnen und in der obigen Weise rechromatographiert. Erhalten wurden 18 mg 1-Hydroxy-Cytorhodin N + 0 mit weniger als 10 % restlichem Cytorhodin N + 0.
C60H88N2O22, M ber., 1188 (FAB-MS bestätigt). - Das isolierte Produkt ist ein 1 : 1 Gemisch der beiden strukturisomeren Komponenten 1-OH-N + 1-0H-0. Dies geht aus dem 1H-NMR-Spektrum, Abbauversuchen (Hydrogenolyse) und Bestimmung der einzelnen Zuckerbausteine nach Hydrogenolyse und Totalhydrolyse hervor.C 60 H 88 N 2 O 22 , M calc., 1188 (FAB-MS confirmed). - The isolated product is a 1: 1 mixture of the two structural isomeric components 1-OH-N + 1-0H-0. This is evident from the 1 H-NMR spectrum, degradation tests (hydrogenolysis) and determination of the individual sugar components after hydrogenolysis and total hydrolysis.
Isolierung von 1-Hydroxy-Cytorhodin P durch "reversed phase"-HPLCIsolation of 1-hydroxy-cytorhodin P by "reversed phase" HPLC
Durch übliche Säulenchromatographie an 31 µ Kieselgel pH 7,5 wurde ein stark violettes Produkt erhalten, dessen Hauptkomponente bei DC (System A) und analytischer HPLC an Kieselgel eine geringfügig höhere Retention als 1-Hydroxy-Cytorhodin C zeigte. Durch wiederholte präparative "reversed phase"-HPLC konnte die neue Verbindung isoliert werden. Dazu wurden z.B. 175 mg des violetten Rohproduktes in 2,5 ml der mobilen Phase CHCl3-Methanol 15 % Ammonacetat in Wasser 500 : 1100 : 300 gelöst, und an ca. 100 g 10u LiChrosorb(R) RP-18 (Merck) in einer Stahlsäule 3,2 x 25 cm unter Druck bei einem Fluß von 4 ml/Min. chromatographiert. Das Eluat wurde in Fraktionen von 8 ml gesammelt und zusammengehörende Fraktionen nach Beurteilung durch analytische "reversed-phase"-HPLC mit Doppelwellenlängendetektion bei 490 und 560 nm vereinigt.A very violet product was obtained by conventional column chromatography on 31 μ silica gel pH 7.5, the main component of which showed a slightly higher retention than 1-hydroxy-cytorhodin C in TLC (system A) and analytical HPLC on silica gel. The new compound was isolated by repeated preparative "reversed phase" HPLC. For this purpose, for example, 175 mg of the violet crude product were dissolved in 2.5 ml of the mobile phase CHCl 3 -methanol 15% ammonium acetate in water 500: 1100: 300, and in about 100 g of 10u LiChrosorb (R) RP-18 (Merck) in a steel column 3.2 x 25 cm under pressure at a flow of 4 ml / min. chromatographed. The eluate was collected in fractions of 8 ml and related fractions were pooled after evaluation by analytical "reversed-phase" HPLC with double wavelength detection at 490 and 560 nm.
C60H82N2O22, M ber, 1182 (FAB-MS bestätigt).C 60 H 82 N 2 O 22 , M ber, 1182 (FAB-MS confirmed).
Stahlsäulen (4,6 x 250 mm), gepackt mit 7 u LiChrosorb(R) Si 60 (Merck) oder 7 µ Kieselgel 60 (Grace); mobile Phase: CHCl3-Methanol-86% ige Essigsäure-Triäthylamin-Wasser 80 : 10 : 10 : 2 : 0,01 Fluß: 0,5 - 1,5 ml/Min.; Nachweis bei 260 oder 490 nm mit Durchflußphotometer.Steel columns (4.6 x 250 mm) packed with 7 u LiChrosorb (R) Si 60 (Merck) or 7 µ silica gel 60 ( G race); mobile phase: CHCl 3 -methanol-86% acetic acid-triethylamine-water 80: 10: 10: 2: 0.01 flow: 0.5 - 1.5 ml / min .; Detection at 260 or 490 nm with flow photometer.
Stahlsäulen (4,6 x 250 mm) gepackt mit 14 µ Lichrosorb(R) RP-18 (Merck)Steel columns (4.6 x 250 mm) packed with 14 µ Lichrosorb (R) RP-18 (Merck)
mobile Phase: CHC13-Methanol-10 % Ammonacetat in Wasser 250 : 1050 : 375mobile phase: CHC1 3 -methanol-10% ammonium acetate in water 250: 1050: 375
Fluß: 0,5 - 1,5 ml/Min.; Nachweis bei 490 nm und 560 nm mit Durchflußphotometer.Flow: 0.5-1.5 ml / min .; Detection at 490 nm and 560 nm with flow photometer.
Die verwendeten analytischen und präparativen HPLC-Säulen wurden i.a. mit den von der Fa. Merck (Darmstadt) empfohlenen Verfahren mit Hilfe einer pneumatischen Pumpe (Haskel) selbst gefüllt.The analytical and preparative HPLC columns used were generally filled with the methods recommended by Merck (Darmstadt) using a pneumatic pump (Haskel).
Zur üblichen präparativen Säulenchromatographie wurde 319 Kieselgel 60 (Gracc) oder 15-40 µ Kieselgel 60 (Merck) in offenen Glassäulen oder in unter Druck gepackten Stahlsäulen verwendet. Als mobile Phase dienten Lösungsmittelgemische ähnlicher Zusammensetzung wie das o.a. analytische HPLC System gegebenenfalls mit herabgesetzten Anteilen an Methanol, Essigsäure und Wasser.For the usual preparative column chromatography, 31 9 silica gel 60 (Gracc) or 15-40 μ silica gel 60 (Merck) were used in open glass columns or in steel columns packed under pressure. Solvent mixtures of a similar composition to the above-mentioned analytical HPLC system, optionally with reduced proportions of methanol, acetic acid and water, served as the mobile phase.
Zur Chromatographie v.a. starker polarer Komponenten wurde 31 µ Kieselgel 60 (Gracc) mit 2 N HC1 metallfrei gewaschen und nach Auswaschen der Säure in wäßriger Suspension mit 5 N NaOH behandelt, bis sich ein pH von 7.5 eingestellt hatte. Nach Dekantieren wurde das modifizierte Kieselgel bei 130°C getrocknet, gesiebt und mit der mobilen Phase in die Säule eingeschlämmt. Als mobile Phase wurden Lösungsmittelgemische CHCl3 / Wasser / Methanol 13 : 4 : 3-7 eingesetzt. Das Belastungsverhältnis betrug etwa 1 : 100 - 1 : 300.For the chromatography of strong polar components in particular, 31 μ of silica gel 60 (Gracc) was washed with 2 N HCl 1 free of metal and, after washing out the acid in aqueous suspension, treated with 5 N NaOH until a pH of 7.5 had been established. After decanting, the modified silica gel was dried at 130 ° C., sieved and slurried into the column with the mobile phase. Solvent mixtures CHCl 3 / water / methanol 13: 4: 3-7 were used as the mobile phase. The load ratio was about 1: 100 - 1: 300.
Kieselgelplatten F254 (Merck) wurden entwickelt mit System A: CHCl3-Methanol-96 % Essigsäure-Wasser-Triäthylamin 80 : 10 : 10 : 2 : 0,01 oder System B: CHCl3-Methanol-99%ige Essigsäure 75 : 15 : 10 : 2.Silica gel plates F 254 (Merck) were developed with System A: CHCl 3 -Methanol-96% acetic acid-water-triethylamine 80: 10: 10: 2: 0.01 or System B: CHCl 3 -Methanol-99% acetic acid 75: 15: 10: 2.
Die übliche Aufarbeitung wurde wie folgt ausgeführt: vereinigte Fraktionen aus "reversed phase"-HPLC, wurden mit etwa der Hälfte ihres Volumens an Wasser versetzt und anschließend Chloroform bis zur Phasentrennung zugegeben. Die abgetrennte Chloroformphase wurde mit Wasser gewaschen, über wasserfreiem Na2SO4 getrocknet und i.V. eingedampft. Die so erhaltenen Produkte wurden auf folgende Weise von begleitenden Fetten, Weichmachern und Metallionen befreit: das Produkt - z.B. 30 mg - wurden in 20 ml Natrium-Acetatpuffer, pH 3,5 gelöst, mit 1 ml 0,001 M wäßriger Äthylendiamintetraessigsäure (EDTA; mit NaOH auf pH 3,5 eingestellt) versetzt, und mit 5 ml Toluol geschüttelt. Die Toluolphase wurde verworfen, die wäßrige Phase mit 2 N NaOH auf pH 7,5 eingestellt und mit 20 ml CHC13 unter gutem Schütteln extrahiert. Nach dem Trocknen über wasserfreiem Na2S04 wurde filtriert und im Vakuum eingedampft. Gegebenenfalls wurde der Rückstand in wenig CHC13 gelöst, die Lösung durch eine Glasfritte abgesaugt und nach Zusatz von Heptan bis zur Trübung i. V. eingedampft.The usual work-up was carried out as follows: combined fractions from "reversed phase" HPLC were mixed with about half their volume in water and then chloroform was added until the phases separated. The separated chloroform phase was washed with water, dried over anhydrous Na 2 SO 4 and evaporated in vacuo. The products thus obtained were freed of accompanying fats, plasticizers and metal ions in the following way: the product - for example 30 mg - was dissolved in 20 ml of sodium acetate buffer, pH 3.5, with 1 ml of 0.001 M aqueous ethylenediaminetetraacetic acid (EDTA; with NaOH adjusted to pH 3.5), and shaken with 5 ml of toluene. The toluene phase was discarded, the aqueous phase was adjusted to pH 7.5 with 2N NaOH and extracted with 20 ml of CHC1 3 with good shaking. After drying over anhydrous Na 2 S0 4 , the mixture was filtered and evaporated in vacuo. If necessary, the residue was dissolved in a little CHC1 3 , the solution was suctioned off through a glass frit and, after adding heptane, i until turbid. V. evaporated.
Die Identifizierung der Komponenten in den vorstehenden Beispielen erfolgte unter den anschließend beschriebenen Meßbedingungen:The components in the above examples were identified under the measurement conditions described below:
Die Protonen-Resonanzspektren (lH-NMR-Spektren) wurden auf einen HX-270 BRUKER Fourier-Transform Kernresonanzspektrometer bei 270 MHZ gemessen. Die Konzentrationen betrugen 2 - 4 mg/0,5 ml 99,8 % CDCl3; die Lösungen wurden sofort nach Herstellung mit 0,1 ml 5% iger Na2CO3 in 99,5 % D20 geschüttelt.The proton resonance spectra ( 1 H-NMR spectra) were recorded on a HX-270 BRUKER Fourier transform nuclear magnetic resonance spectrometer measured at 270 MHz. The concentrations were 2-4 mg / 0.5 ml 99.8% CDCl 3 ; the solutions were shaken immediately after preparation with 0.1 ml of 5% Na 2 CO 3 in 99.5
Die in den Abbildungen mit einem Stern versehenen Signale rühren her von niedermolekularen Verunreinigungen im %o-Bereich und von Lösungsmittelresten.The signals marked with an asterisk in the figures derive from low-molecular impurities in the% o range and from solvent residues.
Die Massenspektren wurden auf dem Massenspektrometer MS-902 S, AEI, unter Verwendung einer FAB-(Fast-Atom-Bombardment-) Ionenquelle gemessen. Die Substanzen wurden in einer Matrix von Thioglycerin in die Ionenquelle eingebracht, teilweise unter Zusatz von Ammoniumchlorid.Mass spectra were measured on the MS-902 S, AEI mass spectrometer using an FAB (Fast Atom Bombardment) ion source. The substances were introduced into the ion source in a matrix of thioglycerin, sometimes with the addition of ammonium chloride.
Die Absorptionsspektren wurden im Bereich 200 - 700 nm gemessen in:
- a) Wasser-Methanol 1 : 9
- b) 10 % 1 n HC1 in Methanol
- c) 10 % 1 n NaOH in Methanol.
- a) Water-methanol 1: 9
- b) 10% 1N HC1 in methanol
- c) 10% 1N NaOH in methanol.
Die Substanzkonzentration betrug 10 - 30 mg/l; angegeben werden die Absorptionsmaxima in nm und die molaren Extinktionskoeffizienten (log ε).The substance concentration was 10 - 30 mg / l; the absorption maxima are given in nm and the molar extinction coefficients (log ε).
Die Bestimmung der zytostatischen Wirksamkeit der hier beschriebenen Verbindungen erfolgte an L1210 Leukämiezellen der Maus. Im einzelnen wurden folgende Testsysteme verwendet:The cytostatic activity of the compounds described here was determined on L1210 mouse leukemia cells. The following test systems were used:
Bei dieser Methode wird in vitro nach Inkubation der Zellen mit unterschiedlichen Konzentrationen der Testsubstanz ermittelt, inwieweit die Zellen radioaktive DNA-Vorläufer (z.B. C14 markiertes Thymidin) einbauen können. Unbehandelte L1210 Zellen werden den gleichen Testbedingungen unterworfen und dienen als Kontrolle. Im folgenden ist die Methode kurz beschrieben.In this method, the cells are incubated with different concentrations of in vitro after incubation Test substance determines the extent to which the cells can incorporate radioactive DNA precursors (eg C14-labeled thymidine). Untreated L1210 cells are subjected to the same test conditions and serve as a control. The method is briefly described below.
L1210 Zellen in exponentieller Wachstumsphase (5 x 10 3/ml in RPMI 1640) werden in einer Mikrotiterplatte 72 Stunden mit unterschiedlichen Konzentrationen der Testsubstanz inkubiert (37°C, 5 % C021 95 % relative Luftfeuchte). Kontrolle bestehen aus Zellen, die lediglich mit frischem Medium inkubiert werden. Alle Bestimmungen werden als 4fach Bestimmungen durchgeführt. Nach 65 Stunden werden 50 µl C-1 14 Thymidin (1,5 µc/ml) zugegeben, um die DNA der Zelle radioaktiv zu markieren. Nach 7 Stunden Inkubation werden die Zellen abgesaugt, die DNA mit 5%iger Trichloressigsäure gefällt und nacheinander mit Wasser bzw. Methanol gewaschen.L1210 cells in exponential growth phase (5 x 10 3 / ml in RPMI 1640) are incubated in a microtiter plate for 72 hours with different concentrations of the test substance (37 ° C, 5% C0 21 95% relative air humidity). Controls consist of cells that are only incubated with fresh medium. All determinations are carried out as 4-fold determinations. After 65 hours, 50 μl of C-1 14 thymidine (1.5 μc / ml) are added in order to radioactively label the DNA of the cell. After 7 hours of incubation, the cells are suctioned off, the DNA is precipitated with 5% trichloroacetic acid and washed successively with water or methanol.
Nach Trocknung bei 50°C wird die in die DNA eingebaute Radioaktivität nach Zugabe von 5 ml Szintillationsflüssigkeit ermittelt.After drying at 50 ° C., the radioactivity built into the DNA is determined after adding 5 ml of scintillation fluid.
Die Ergebnisse werden angegeben als Verhältnis des Szintillationsindex nach Inkubation mit der Testsubstanz in Prozent der unbehandelten Kontrolle. Aus den so erhaltenen Meßwerten wird die Dosiswirkungskurve ermitelt und graphisch die IC50, d.h. die Konzentration, die unter Testbedingungen den Einbau von radioaktivem Thymidin um 50 % gegenüber der Kontrolle erniedrigt, ermittelt. Die IC50 Werte der hier beschriebenen Verbindungen im Vergleich zu Adriamycin (ADM) werden in der Tabelle 1 zusammengefaßt.The results are given as the ratio of the scintillation index after incubation with the test substance in percent of the untreated control. The dose-response curve is determined from the measured values thus obtained and the IC 50 , ie the concentration which reduces the incorporation of radioactive thymidine by 50% compared to the control under test conditions, is determined graphically. The IC 50 values of the compounds described here in comparison to adriamycin (ADM) are summarized in Table 1.
Diese Methode dient zum Nachweis eines Einflusses der Testsubstanzen auf das Wachstumsverhalten der Zellen über mehrere Generationen (bei einer Zellzykluszeit von 10-12 Stunden werden in der Testzeit von 7 Tagen ca. 14 aufeinanderfolgende Generationen beobachtet).This method serves to demonstrate the influence of the test substances on the growth behavior of the cells over several generations (with a cell cycle time of 10-12 hours, approximately 14 consecutive generations are observed in the test time of 7 days).
Zytostatisch wirksame Substanzen bewirken in diesem Test eine Reduktion der zu beobachtenden Koloniezahl gegenüber einer unbehandelten Kontrolle. Im einzelnen wird der Test wie folgt durchgeführt:In this test, cytostatic substances reduce the number of colonies to be observed compared to an untreated control. The test is carried out as follows:
500 Leukämiezellen pro Platte werden mit unterschiedlichen Konzentrationen von Testsubstanz 1 Stunde bei 37°C inkubiert. Anschließend werden die Zellen zweimal mit McCoy5A Medium gewaschen und schließlich in Petrischalen nach Zusatz von 0,3 % Agar ausgegossen. Kontrollen werden lediglich mit frischem Medium inkubiert. Anstelle der lstündigen Inkubation werden in manchen Fällen unterschiedliche Konzentrationen und Testsubstanz der oberen Agarschicht zugemischt, um so eine kontinuierliche Exposition der Zellen über die gesamte Inkubationszeit zu erreichen. Nach Erstarren des Agars werden die Platten im Brutschrank 7 Tage bei 37°C inkubiert (5 % C02, 95 % relative Luftfeuchtigkeit). Anschließend wird die Anzahl der entstandenen Kolonien mit einem Durchmesser 60 p gezählt. Die Ergebnisse werden angegeben als Koloniezahl in behandelten Agarplatten in Prozent der unbehandelten Kontrolle. Aus der so erhaltenen Dosiswirkungskurve wird die IC50 als Maß für die Wirksamkeit der Substanz ermittelt. Die Ergebnisse für die hier beschriebenen Verbindungen im Vergleich zu Adriamycin sind in der Tabelle 1 zusammengefaßt.
Wie vorstehend ausgeführt, besitzen die erfindungsgemäßen Verbindungen zytostatische Wirksamkeit, d.h. therapeutische Wirkung gegen Tumoren, insbesondere malign.e Tumoren bei Tieren und Menschen.As stated above, the compounds of the invention have cytostatic activity, i.e. therapeutic effect against tumors, especially malignant tumors in animals and humans.
Die Verbindungen und die Säureadditionssalze können daher als Medikamente zur Behandlung von Tumoren verwendet werden. Die Verbindungen können auf verschiedene Weise in Abhängigkei von der Dosierungsform verabreicht werden. Normalerweise werden die Verbindungen mit pharmazeutisch üblichen Trägerstoffen oder Verdünnungsmitteln vermischt verabreicht. So können sie z.B. einzeln oder in Mischung zusammen mit Trägerstoffen wie Maltose oder Lactose oder als nichttoxische Komplexe, z.B. als Desoxyribonukleinsäure-Komplex verabreicht werden.The compounds and the acid addition salts can therefore be used as drugs for the treatment of tumors. The compounds can be administered in various ways depending on the dosage form. The compounds are normally administered mixed with standard pharmaceutical carriers or diluents. So you can e.g. individually or in a mixture together with carriers such as maltose or lactose or as non-toxic complexes, e.g. be administered as a deoxyribonucleic acid complex.
Eine typische Verabreichungsart ist die Injektion einer Lösung der erfindungsgemäßen Verbindungen in destilliertem Wasser oder in physiologischer Kochsalzlösung. Die Lösungen können intraperitoneal, intravenös oder intraarteriell injiziert werden.A typical mode of administration is the injection of a solution of the compounds according to the invention in distilled water or in physiological saline. The solutions can be injected intraperitoneally, intravenously or intraarterially.
Tagesdosis und Einheitsdosis können aus Tierversuchen und auch aus in vitro-Tests in der Weise festgesetzt werden, daß die Gesamtdosis, die kontinuierlich oder in Abständen verabreicht wird, einenvorher festgelegten Bereich nicht überschreitet. So beträgt die Gesamtdosis für einen Behandlungszyklus etwa 0,5-5 mg/kg Körpergewicht. Diese Dosis kann in entsprechenden Bruchteilen über einen Zeitraum von 7 Tagen verabreicht werden. Es ist jedoch klar, daß konkrete Dosen für die Behandlung von Mensch oder Tier individuell festgelegt werden können in Abhängigkeit von der jeweiligen Situation, z.B. Alter, Körpergewicht, Geschlecht, Empfindlichkeit, Nahrung, Zeitpunkt der Verabreichung, weiterenverabreichten Medikamenten, körperlichem Zustand der Patienten und Schwere der Erkrankung.Daily dose and unit dose can be determined from animal experiments and also from in vitro tests in such a way that the total dose, which is administered continuously or at intervals, does not exceed a predetermined range. So the total dose for one treatment is cycle about 0.5-5 mg / kg body weight. This dose can be administered in appropriate fractions over a period of 7 days. However, it is clear that specific doses for the treatment of humans or animals can be determined individually depending on the particular situation, for example age, body weight, gender, sensitivity, food, time of administration, further medication administered, patient's physical condition and severity the disease.
Claims (15)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT85108137T ATE46701T1 (en) | 1984-07-10 | 1985-07-01 | 1-HYDROXY-CYTORHODINE, A MICROBIOLOGICAL PROCESS FOR THEIR PRODUCTION AND ITS USE AS CYTOSTATICS. |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3425357 | 1984-07-10 | ||
| DE19843425357 DE3425357A1 (en) | 1984-07-10 | 1984-07-10 | 1-HYDROXY-CYTORHODINE, A MICROBIOLOGICAL METHOD FOR THE PRODUCTION THEREOF AND THEIR USE AS CYTOSTATICS |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| EP0167954A2 true EP0167954A2 (en) | 1986-01-15 |
| EP0167954A3 EP0167954A3 (en) | 1986-11-20 |
| EP0167954B1 EP0167954B1 (en) | 1989-09-27 |
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ID=6240265
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| Application Number | Title | Priority Date | Filing Date |
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| EP85108137A Expired EP0167954B1 (en) | 1984-07-10 | 1985-07-01 | 1-hydroxy-cytorhodines, a microbiological method for their preparation and their use as cytostatic agents |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0167954B1 (en) |
| JP (1) | JPS6144841A (en) |
| KR (1) | KR860001190A (en) |
| AT (1) | ATE46701T1 (en) |
| AU (1) | AU586977B2 (en) |
| DE (2) | DE3425357A1 (en) |
| DK (1) | DK313985A (en) |
| ES (1) | ES8609356A1 (en) |
| FI (1) | FI81608C (en) |
| GR (1) | GR851682B (en) |
| HU (1) | HUT42134A (en) |
| IL (1) | IL75748A0 (en) |
| NO (1) | NO852758L (en) |
| PT (1) | PT80790B (en) |
| ZA (1) | ZA855154B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0203329A1 (en) * | 1985-04-12 | 1986-12-03 | Sanraku Incorporated | Obelmycin A |
| EP0242695A1 (en) * | 1986-04-08 | 1987-10-28 | Sanraku Incorporated | New anthracycline antibiotics DCP-1 and 2 |
| RU2132185C1 (en) * | 1998-06-15 | 1999-06-27 | Саратовский государственный университет им.Н.Г.Чернышевского | Agent for treatment and prophylaxis of intestine helminthism in animals |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3709337A1 (en) * | 1987-03-21 | 1988-10-13 | Hoechst Ag | NEW ANTHRACYCLINE GLYCOSIDES, A METHOD FOR THE PRODUCTION THEREOF AND THEIR USE AS CYTOSTATICS |
| DE3712350A1 (en) * | 1987-04-11 | 1988-10-20 | Behringwerke Ag | SEMISYNTHETIC RHODOMYCINES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE AS CYTOSTATICS |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD122069A1 (en) * | 1974-04-29 | 1976-09-12 | ||
| JPS6023679B2 (en) * | 1979-07-13 | 1985-06-08 | メルシャン株式会社 | Rhodomycin group antibiotics and their production method |
| EP0050724B1 (en) * | 1980-10-27 | 1985-01-09 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Process for anthracycline glycosides |
| DE3325957A1 (en) * | 1983-07-19 | 1985-02-07 | Hoechst Ag, 6230 Frankfurt | ANTHRACYCLINE DERIVATIVES, A METHOD FOR THE PRODUCTION THEREOF AND THEIR USE AS A CYTOSTATIKA |
| JPS61236792A (en) * | 1985-04-12 | 1986-10-22 | Sanraku Inc | Novel anthracycline antibiotic |
-
1984
- 1984-07-10 DE DE19843425357 patent/DE3425357A1/en not_active Withdrawn
-
1985
- 1985-07-01 DE DE8585108137T patent/DE3573263D1/en not_active Expired
- 1985-07-01 EP EP85108137A patent/EP0167954B1/en not_active Expired
- 1985-07-01 AT AT85108137T patent/ATE46701T1/en not_active IP Right Cessation
- 1985-07-04 HU HU852605A patent/HUT42134A/en unknown
- 1985-07-08 FI FI852696A patent/FI81608C/en not_active IP Right Cessation
- 1985-07-08 ES ES544960A patent/ES8609356A1/en not_active Expired
- 1985-07-08 GR GR851682A patent/GR851682B/el unknown
- 1985-07-09 NO NO852758A patent/NO852758L/en unknown
- 1985-07-09 PT PT80790A patent/PT80790B/en not_active IP Right Cessation
- 1985-07-09 AU AU44734/85A patent/AU586977B2/en not_active Ceased
- 1985-07-09 IL IL75748A patent/IL75748A0/en unknown
- 1985-07-09 JP JP60149404A patent/JPS6144841A/en active Pending
- 1985-07-09 ZA ZA855154A patent/ZA855154B/en unknown
- 1985-07-09 DK DK313985A patent/DK313985A/en not_active Application Discontinuation
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0203329A1 (en) * | 1985-04-12 | 1986-12-03 | Sanraku Incorporated | Obelmycin A |
| EP0242695A1 (en) * | 1986-04-08 | 1987-10-28 | Sanraku Incorporated | New anthracycline antibiotics DCP-1 and 2 |
| RU2132185C1 (en) * | 1998-06-15 | 1999-06-27 | Саратовский государственный университет им.Н.Г.Чернышевского | Agent for treatment and prophylaxis of intestine helminthism in animals |
Also Published As
| Publication number | Publication date |
|---|---|
| AU4473485A (en) | 1986-01-16 |
| ATE46701T1 (en) | 1989-10-15 |
| AU586977B2 (en) | 1989-08-03 |
| ZA855154B (en) | 1986-02-26 |
| ES544960A0 (en) | 1986-08-01 |
| FI81608B (en) | 1990-07-31 |
| HUT42134A (en) | 1987-06-29 |
| EP0167954A3 (en) | 1986-11-20 |
| ES8609356A1 (en) | 1986-08-01 |
| FI852696L (en) | 1986-01-11 |
| EP0167954B1 (en) | 1989-09-27 |
| PT80790B (en) | 1987-10-20 |
| FI852696A0 (en) | 1985-07-08 |
| GR851682B (en) | 1985-11-26 |
| DE3573263D1 (en) | 1989-11-02 |
| DK313985D0 (en) | 1985-07-09 |
| IL75748A0 (en) | 1985-11-29 |
| FI81608C (en) | 1990-11-12 |
| DK313985A (en) | 1986-01-11 |
| KR860001190A (en) | 1986-02-24 |
| PT80790A (en) | 1985-08-01 |
| JPS6144841A (en) | 1986-03-04 |
| NO852758L (en) | 1986-01-13 |
| DE3425357A1 (en) | 1986-01-23 |
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